This invention relates to the identification of potential fungicides and insecticides using a screening method that reverses the calcofluor inhibition of chitin biosynthesis in yeast.
The polysaccharide chitin is a structural cell wall component of all fungi except some Basidiomycetes fungi and most Oomycetes, and is the most abundant organic skeletal component of invertebrates, making up, for example, from about 25 to 60% of the dry weight of insect cuticles. Chitin consists primarily of linear polymers of the amino sugar N-acetyl-D-glucosamine joined in 1,4-xcex2-glucosidic linkage. Thus, chitin bears a close resemblance to cellulose, the major structural polysaccharide of plants; indeed, the only chemical difference between them is that in chitin the hydroxyl group on the 2-position is an acetoamido group instead of an hydroxyl. However, because of its widespread occurrence in fungi and arthropods, the total world-wide production of chitin vastly exceeds cellulose (Neville, A. C., The Biology of the Arthropod Cuticle, Springer-Verlag, New York, 1975, pages 71 to 76).
Many fungi and arthropods having chitinous cell walls or exoskeletons are injurious to plants and animals, causing a legion number of diseases. To name but a few, fungal species containing chitin cause wheat eyespot, rice sheath blight, damping off, apple scab, pepper botrytis, rice blast, sugar beet cercospora, tomato early blight, wheat leaf rust, and wheat powdery mildew. Fungal species also cause myriad cutaneous and systemic mycoses in human beings and other animals, including candidiasis, histoplasmosis, blastomycosis, sporotrichosis, cryptococcosis, and the like. Insects are vectors of viruses causing arboviral encephalitides, yellow fever, and dengue, protozoa causing malarias, trypanosomiases, and leishmaniases, and various harmful helminths. Crustaceans carry some infectious helminths and trematodes.
Most fungicides and insecticides that are used to control or cure these diseases by killing or controlling their causative agents, intermediate hosts, or vectors employ various modes of action including physical poisons that suffocate or dessicate organisms; protoplasmic poisons such as arsenicals that kill by precipitating or deactivating proteins, enzymes or other cellular constituents; respiratory poisons that deactivate respiratory enzymes; and various poisons that affect different tissue systems such as tubules or nerves. Of course, preferred agents do not injure the host plant or animal, and most preferably have no effect whatsoever on the host. Because of the complexity and interdependence of life processes, however, this goal is not always achieved, so that many fungicides and insecticides exhibit some toxicity to the host. Others cause unexpected side effects.
Since chitin is not a usual constituent of most plants and vertebrates, chitin biosynthesis inhibitors can be employed as selective antifungal and/or insecticide agents. Applied to ornamental or edible plants or animals, these offer the advantage of targeting undesirable fungi or insects without harming significantly the host plant or vertebrate animal. 1-(2,6-Dichlorobenzoyl)-3-(3,4-dichlorophenyl)urea, for example, has been suggested as a chitin-inhibiting insecticide (Neville, cited above). Antifungals that inhibit chitin synthesis include nikkomycin and polyoxin D.
Calcofluor white is a fluorescent brightener used commercially to whiten textiles and paper. The fluorochrome has been used as a stain for cell wall materials in fungi, algae, and higher plants. It can exhibit antifungal properties, binding to nascent chitin microfibrils in fungal cell walls containing chitin. Exposure of yeast (Saccharomyces cerevisiae) to calcofluor white, for example, induces abnormally thick walls between mother and daughter cells during cell division as a result of the massive deposition of anomalous crystallized chitin. The dye interaction appears to enhance the rate of chitin polymerization, producing levels of chitin that are inhibitory to cell growth and viability (Roncero, C., et al., J. Bact. 170: 1945-1949 (1988)). Microscopic examination of calcofluor-inhibited cells reveals high levels of chitin deposition. This putative mechanism for calcofluor white action is supported by the observation that mutants selected for calcofluor white resistance show decreased levels of chitin synthesis (Roncero, C., et al., J. Bact. 170: 1950-1954 (1988)).
The interaction of calcofluor white with growing S. cerevisiae cells generally requires a pH of above 4 and close to 6 or 6.5 (Roncero, et al., cited above at 1946). This can perhaps be understood in view of the fact that a major chitinase in yeast is active only under acidic conditions (Correa, J., et al., J. Biol. Chem. 257: 1392-1397 (1982)). It is possible that this enzyme opposes the action of calcofluor, and thus the activity of calcofluor is augmented under conditions of reduced chitinase activity.
It is an object of the invention to provide a screening test for the identification of agents exhibiting potential fungicidal and insecticidal activity for a wide variety of agricultural, medical, and veterinary uses.
It is a further and more specific object of the invention to identify agents that inhibit chitin biosynthesis.
These and other objects are accomplished by the present invention, which provides a method for the identification of agents which inhibit chitin synthesis, and thus possess fungicidal and insecticidal activity making them potentially suitable as selective fungicides or insecticides. The method is a screening test whereby test samples are incubated in a fungal culture with calcofluor white. Agents exhibiting potentially desirable fungicidal or insecticidal properties inhibit chitin synthesis, and, by doing so, reverse calcofluor white inhibition of the culture. Agents that are positive in the test produce enhanced fungus growth because they rescue the growing fungus from the adverse effects of calcofluor white.
In the practice of this inventive method for screening for the presence or absence of chitin synthesis inhibition by a test sample, the test sample is added to a chitin-producing fungus, e.g., a yeast, culture or culture area containing calcofluor white. The culture is incubated with the test sample for such time under such conditions sufficient to observe yeast cell growth inhibition in a corresponding culture containing calcofluor but no test sample. The extent of growth in the culture or culture area containing test sample is then compared with the extent of growth in the culture or culture area containing no test sample. The presence of chitin synthesis inhibition is determined by observation of whether culture growth in the presence of test sample exceeds growth in its absence.
In a preferred screening test, a Saccharomyces cerevisiae strain exhibiting little or no calcofluor white resistance is grown in culture at neutral pH in the presence of calcofluor white and test samples. Potentially active agents are identified by the observation of enhanced growth of the cultured yeast. In especially preferred embodiments, a positive control is employed to assist in the identification of potential agents. In these embodiments, a known chitin sythesis inhibitor such as nikkomycin Z is added to the culture or culture area, and this is compared to the culture with the test sample.
In a particularly preferred embodiment, the yeast is grown in a solidified media in the presence of calcofluor in a plate or dish, so that test samples and positive controls can be observed visually and simultaneously as regions of the same culture. Actives produce a turbid zone of growth around the test sample in the lawn of the culture.
This invention is based upon the finding that chemical and biochemical agents of potential value as fungicides or insecticides are identified in Saccharomyces cerevisiae cultures containing calcofluor white, a fluorochrome that causes lethal chitin polymerization in the yeast. Cultures containing test samples that inhibit chitin formation exhibit enhanced growth because the test sample rescues the cultured yeast cells from the deleterious effects of calcofluor white.
In the practice of this invention, test samples are incubated in the presence of calcofluor white in cultures of any fungal species that produce chitin, such as unicellular fungi. A preferred method employs common baker""s yeast, Saccharomyces cerevisiae, because it is readily available and easy to culture. A known chitin synthesis inhibitor is employed as a positive control. Potentially active agents are identified by the observation of enhanced yeast growth.
A preferred method comprises adding a test sample to a Saccharomyces cerevisiae culture containing calcofluor white or a calcofluor white derivative. The test sample is introduced to a disk or a well on a culture plate in a standard diffusion assay using solidified media, or introduced into one of a series of equivalent tissue culture tubes or bottles in a standard turbidity assay using liquid media. The culture is incubated for such time under such conditions sufficient to observe yeast cell growth inhibition in a corresponding culture or culture plate area containing calcofluor white but no test sample. The extent of growth of the culture containing or surrounding the test sample is compared with the extent of growth in the culture or culture area containing no test sample. The presence of chitin synthesis inhibition is determined by observing whether growth in the presence of test sample exceeds growth in its absence. In a culture plate, this is a turbid zone of growth surrounding the test sample. In a culture tube series, this is enhanced turbidity.
Preferred methods employ a known chitin synthesis inhibitor such as nikkomycin Z as a positive control. The control is useful in discerning whether the screen is functioning properly and in identifying positives by direct comparison. In a disk or well diffusion assay, the control is introduced to a disk or a well in the culture plate at the same time the test sample is introduced. After the incubation period, growth in the vicinity of the control exceeds growth in the culture where there is no test sample. A test sample that inhibits chitin biosynthesis will exceed growth in the rest of the culture even if it does not approximate or exceed growth in the vicinity of the positive control. Likewise, in a turbidity assay, the control is introduced into an equivalent tissue culture tube or bottle and incubated with test and negative cultures; after incubation, cultures containing a test sample that inhibits chitin biosynthesis exhibit more turbidity than that observed in tubes containing yeast and calcofluor white only, exhibiting enhanced turbidity analogous to that observed with the positive control.
By xe2x80x9ccalcofluor whitexe2x80x9d is meant the fluorescent brightener (2,2xe2x80x2-(1,2-ethenediyl)bis[5-[[4-bis (2-hydroxyethyl) amino)-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-benzenesulfonic acid or 4,4xe2x80x2-bis[4-anilino-bis(xcex2-hydroxyethyl) amino-s-triazine-2-ylamino]-2,2xe2x80x2-stilbene disulfonic acid) and its derivatives such as its esters and salts and includes its benzidine derivative, Congo red (3,3xe2x80x2-[[1,1xe2x80x2biphenyl]-4,4xe2x80x2-diylbis(azo)]bis(4-amino-1-naphthalenesulfonicacid]), which has also been shown to activate chitin polymerization in fungal cell walls (Roncero, C., and Duran, A., J. Bact. 163: 1180-1185 (1985)). Preferred embodiments employ the disodium salt of calcofluor white, which is commercially available, marketed under the names Calcofluor White M2R, Tinopal LPW, and C.I. 40622.
Calcofluor white is added to Saccharomyces cerevisiae cultures in preferred embodiments. Many readily available strains of S. cerevisiae may be employed. Strains showing little or no calcofluor white resistance are preferred, especially those sensitive to calcofluor white. Wild-type strains are especially preferred in some embodiments. Typical S. cerevisiae strains include, but are not limited to, wild-type strain A.T.C.C. 12341, X2180 (A.T.C.C. 26109), or X2180-1A obtained from the Yeast Genetic Stock Center.
Any type of solidified or liquid media that will support growth and reproduction of S. cerevisiae may be employed as cultures in the method of this invention. Numerous yeast media are known to the skilled artisan, and include, for example, yeast basal growth media (YBGM) containing glucose, vitamins, minerals, and water. Preferred media are solidified by adding agar or gelatin; especially preferred are media solidified with agar. Preferred media are buffered and neutral, i.e., have a pH of about 4 to about 8, preferably about 5.5 to about 7.5. In some embodiments, the preferred pH of the media ranges from about 6 to about 7.
Enhanced growth in solidified cultures is ordinarily observed visually as turbid areas of growth around disks or wells in the culture plate. Enhanced growth in liquid cultures is observed visually, but is ordinarily determined spectrophotometrically as enhanced optical density (OD) at about 550 to 650 nm.
A distinct advantage of the invention is its speed and simplicity. Baker""s yeast is readily available and inexpensive. Using solidified media in culture plates, the protocol is extremely simple. Many samples can be readily analyzed in a short time.
It is another advantage of the invention that only small amounts of biochemical or chemical agents are required in the test. In a standard assay, for example, which employs solidified media in a plate, as little as 20 xcexcg of a biochemical or chemical test sample can be applied to a disk or in a well. As a positive control, as little as 2 to 10 xcexcg of nikkomycin Z can be used. For fermentation broths, little or no concentration is necessary, but may be required for some samples.
It is a further advantage of the invention that the calcofluor rescue assay is a low positive rate assay ( less than 0.02%), so that secondary tests are not of crucial importance. For an identification of a specific mechanism of action for a newly discovered positive test sample, however, it is advantageous to have more than one biological assay method. Moreover, undesired false positives in the calcofluor rescue screen such as those caused by artifacts of compound-calcofluor complexing are avoided by retesting positives obtained in the primary calcofluor rescue screen using secondary assays which detect in vivo or in vitro inhibition of chitin synthases. Preferred secondary assays differ in action from the primary screen and do not use calcofluor, so they are used to identify chitin synthesis inhibitors that do not act directly on calcofluor. Especially preferred secondary tests assay for chitin synthase inhibition by test samples that inhibit chitin in the primary calcofluor rescue screen. Most especially preferred secondary tests identify the chitin synthase isozyme affected by positive test samples.
One type of calcofluor rescue secondary screen is an in vivo test that assays the effect of compounds on the growth of chitin synthase mutant yeast strains that rely on a single chitin synthase isozyme for survival. A number of these strains have been described, such as the strains that rely on chitin synthase isozymes 2 or 3 described by Shaw, J. A., et al. (J. Cell Biol. 114: 111-123 (1991)) and similar strains described in the Examples section below. In this type of secondary screening test, the test sample added to and incubated in a culture of a mutant yeast strain producing only chitin synthase 2 or 3 (denoted, respectively, Chs2 and Chs3) for such time under such conditions sufficient to observe growth in a corresponding culture containing no test sample. A comparison of the extent of growth in the culture containing test sample with growth in its absence is then made. Inhibition of growth in the presence of test sample indicates inhibition of synthase isozyme produced by the strain. Any type of growth assessment may be employed but, as in the primary calcofluor rescue screen, preferred cultures in the secondary screen are solid, with test samples applied to a disk or well, so that cell growth can be easily determined by visual inspection.
Especially preferred in vivo secondary screens employ a pair of genetically related strains, one producing one isozyme, e.g., Chs2, and the other producing a different isozyme, e.g., Chs3. Using pairs allows for the simultaneous determination that culture conditions are appropriate for growth in the absence of inhibition and the identification of which isozyme may be affected by inhibition. The pairs, for example, include the ECY36-3C and ECY36-3D strains described by Shaw, cited above, that produce only Chs3 and Chs 2, respectively, and similar pairs such as SSY640-10A (containing only Chs3) and SSY-638-3B (containing only Chs 2) described in the Examples below. When nikkomycin Z, an inhibitor of chitin synthase active in the primary screen and known to inhibit the activity of Chs1 to a much greater extent than Chs2 (Cabib, E., Antimicrob. Agents Chemother. 35: 170-173 (1991)) is incubated in these culture pairs, it is found that strains ECY36-3C or SSY640-10A are inhibited, whereas growth of either strain ECY36-3D or SSY638-3B is unaffected by the compound. Thus, nikkomycin Z inhibits Chs3 to a significantly greater extent than Chs2. In a similar manner, a comparison of differential effects observed in the growth of strain pairs in the presence of a test sample allows for the putative identification of which isozyme is affected by the sample.
Alternatively and/or additionally, test samples that are positive in the primary calcofluor rescue screen can be tested in an in vitro enzyme assay for chitin synthase. Any type of chitin synthase assay can be employed, such as uptake of radioactively labelled N-acetylglucosamine chitin subunits, or N-acetylglucosamine precursors or derivatives such as uridine diphospho-N-acetylglucosamine, into chitin (described by Orlean, P., J. Biol. Chem. 262: 5732-5739 (1987)). The assays of this type are generally conducted in the particulate fraction of yeast cells or in isolates of the particulate fraction. Chitin synthase activity is determined after incubation in a buffered assay mixture. The chitin product is quantified by filtration followed by scintillation counting. Detailed descriptions are given hereinafter.
To assist in the identification of the mechanism of action of a test sample that is positive in the primary calcofluor rescue screen, enzyme assays for individual isozymes Chs1, Chs2, and Chs3 are preferred. Isozyme assays employ, as the yeast cells supplying enzyme, mutant strains expressing only one isozyme, such as those listed above for the in vivo assay. Thus, to measure Chs2 activity, chs1, chs3 mutants such as ECY36-3D or SSY638-3B producing Chs2, or a cell that carries a high-copy plasmid with the CHS2 gene such as SSY563-9B described below are used to supply enzyme. Similarly, Chs3 activity is measured using membranes from cells lacking Chs1, and preferably Chs2, such as strains ECY36-3C containing Chs3 or SSY640-10A described below. To assay Chs1 activity, it is sufficient to use a wild-type strain, as this isozyme is the major in vitro chitin synthase. Detailed descriptions are given hereinafter.
Standard in vitro and in vivo fungicide discovery screens are employed as tertiary tests to prioritize actives from the calcofluor rescue screen and the secondary screens. These in vitro screens test samples for their ability to inhibit the growth of selected phytopathogenic fungi cultured in nutrient agar. These include fungi causing wheat eyespot (Pseudocercosporella herpotrichoides), rice sheath blight (Rhizoctonia solani) and damping off (Fusarium oxysporum); all synthesize chitin-containing cell walls. High potency fungicides are active against these species in the 10 ppm range (10 xcexcg/ml), while nikkomycin Z can be detected at xcx9c70 xcexcg/ml in the calcofluor rescue assay if 30 xcexcl volumes are tested in welled plates. Polyoxin D shows excellent activity in the 10 xcexcg/ml concentration range (especially against Rhizoctonia), although nikkomycin Z is only weakly active at 25 xcexcg/ml.
In in vivo screens, a variety of phytopathogenic fungi are used to infect plants treated with test compounds. Active compounds block or reduce the appearance of disease symptoms. A number of model plant infections are employed in the screen and include chitin-containing fungi that cause apple scab (Venturia inaequalis), pepper botrytis (Botrytis cincerea), rice blast (Pyricularia oryzae), sugar beet cercospora (Cercospora beticola), tomato early blight (Alternaria solani), wheat leaf rust (Puccinia recondita tritici), and wheat powdery mildew (Erysiphe graminis tritici). The most potent test compounds in these assays are active in the 10 ppm range.
The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard.